Individualized anti-cancer antibodies

ABSTRACT

The present invention relates to a method for producing patient specific anti-cancer antibodies using a novel paradigm of screening. By segregating the anti-cancer antibodies using cancer cell cytotoxicity as an end point, the process makes possible the production of anti-cancer antibodies customized for the individual patient that can be used for therapeutic and diagnostic purposes. The invention further relates to the process by which the antibodies are made and to their methods of use. The antibodies can be made specifically for one tumor derived from a particular patient and are selected on the basis of their cancer cell cytotoxicity and simultaneous lack of toxicity for non-cancerous cells. The antibodies can be used in aid of staging and diagnosis of a cancer, and can be used to treat tumor metastases. The anti-cancer antibodies can be conjugated to red blood cells obtained from that patient and re-infused for treatment of metastases based upon the recognition that metastatic cancers are usually well vascularized and the delivery of anti-cancer antibodies by red blood cells can have the effect of concentrating the antibodies at the site of the tumor.

REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of application Ser.No. 09/415,278, filed Oct. 8, 1999, now U.S. Pat. No. ______, thecontents of which are herein incorporated by reference.

FIELD OF THE INVENTION

[0002] This invention relates to the production of anti-cancerantibodies customized for the individual patient that can be used fortherapeutic and diagnostic purposes. The invention further relates tothe process by which the antibodies are made and to their methods ofuse.

BACKGROUND OF THE INVENTION

[0003] Each individual who presents with cancer is unique and has acancer that is as different from other cancers as that person'sidentity. Despite this, current therapy treats all patients with thesame type of cancer, at the same stage, in the same way. At least 30% ofthese patients will fail the first line therapy, thus leading to furtherrounds of treatment and the increased probability of treatment failure,metastases, and ultimately, death. A superior approach to treatmentwould be the customization of therapy for the particular individual. Theonly current therapy which lends itself to customization is surgery.Chemotherapy and radiation treatment can not be tailored to the patient,and surgery by itself, in most cases is inadequate for producing cures.

[0004] With the advent of monoclonal antibodies, the possibility ofdeveloping methods for customized therapy became more realistic sinceeach antibody can be directed to a single epitope. Furthermore, it ispossible to produce a combination of antibodies that are directed to theconstellation of epitopes that uniquely define a particular individual'stumor.

[0005] Having recognized that a significant difference between cancerousand normal cells is that cancerous cells contain antigens that arespecific to transformed cells, the scientific community has long heldthat monoclonal antibodies can be designed to specifically targettransformed cells by binding specifically to these cancer antigens; thusgiving rise to the belief that monoclonal antibodies can serve as “MagicBullets” to eliminate cancer cells.

[0006] At the present time, however, the cancer patient usually has fewoptions of treatment. The regimented approach to cancer therapy hasproduced improvements in global survival and morbidity rates. However,to the particular individual, these improved statistics do notnecessarily correlate with an improvement in their personal situation.

[0007] Thus, if a methodology was put forth which enabled thepractitioner to treat each tumor independently of other patients in thesame cohort, this would permit the unique approach of tailoring therapyto just that one person. Such a course of therapy would, ideally,increase the rate of cures, and produce better outcomes, therebysatisfying a long-felt need.

[0008] Historically, the use of polyclonal antibodies has been used withlimited success in the treatment of human cancers. Lymphomas andleukemias have been treated with human plasma, but there were fewprolonged remission or responses. Furthermore, there was a lack ofreproducibility and there was no additional benefit compared tochemotherapy. Solid tumors such as breast cancers, melanomas and renalcell carcinomas have also been treated with human blood, chimpanzeeserum, human plasma and horse serum with correspondingly unpredictableand ineffective results.

[0009] There have been many clinical trials of monoclonal antibodies forsolid tumors. In the 1980s there were at least four clinical trials forhuman breast cancer which produced only one responder from at least 47patients using antibodies against specific antigens or based on tissueselectivity. It was not until 1998 that there was a successful clinicaltrial using a humanized anti-her 2 antibody in combination withCisplatin. In this trial 37 patients were accessed for responses ofwhich about a quarter had a partial response rate and another half hadminor or stable disease progression.

[0010] The clinical trials investigating colorectal cancer involveantibodies against both glycoprotein and glycolipid targets. Antibodiessuch as 17-1A, which has some specificity for adenocarcinomas, hadundergone Phase 2 clinical trials in over 60 patients with only onepatient having a partial response. In other trials, use of 17-1Aproduced only one complete response and two minor responses among 52patients in protocols using additional cyclophosphamide. Other trialsinvolving 17-1A yielded results that were similar. The use of ahumanized murine monoclonal antibody initially approved for imaging alsodid not produce tumor regression. To date there has not been an antibodythat has been effective for colorectal cancer. Likewise there have beenequally poor results for lung cancer, brain cancers, ovarian cancers,pancreatic cancer, prostate cancer, and stomach cancer. There has beensome limited success in the use of anti-GD3 monoclonal antibody formelanoma. Thus, it can be seen that despite successful small animalstudies that are a prerequisite for human clinical trials, theantibodies that have been tested have been for the most partineffective.

Prior Patents

[0011] U.S. Pat. No. 5,750,102 discloses a process wherein cells from apatient's tumor are transfected with MHC genes which may be cloned fromcells or tissue from the patient. These transfected cells are then usedto vaccinate the patient.

[0012] U.S. Pat. No. 4,861,581 discloses a process comprising the stepsof obtaining monoclonal antibodies that are specific to an internalcellular component of neoplastic and normal cells of the mammal but notto external components, labeling the monoclonal antibody, contacting thelabeled antibody with tissue of a mammal that has received therapy tokill neoplastic cells, and determining the effectiveness of therapy bymeasuring the binding of the labeled antibody to the internal cellularcomponent of the degenerating neoplastic cells. In preparing antibodiesdirected to human intracellular antigens, the patentee recognizes thatmalignant cells represent a convenient source of such antigens.

[0013] U.S. Pat. No. 5,171,665 provides a novel antibody and method forits production. Specifically, the patent teaches formation of amonoclonal antibody which has the property of binding strongly to aprotein antigen associated with human tumors, e.g. those of the colonand lung, while binding to normal cells to a much lesser degree. U.S.Pat. No. 5,484,596 provides a method of cancer therapy comprisingsurgically removing tumor tissue from a human cancer patient, treatingthe tumor tissue to obtain tumor cells, irradiating the tumor cells tobe viable but non-tumorigenic, and using these cells to prepare avaccine for the patient capable of inhibiting recurrence of the primarytumor while simultaneously inhibiting metastases. The patent teaches thedevelopment of monoclonal antibodies which are reactive with surfaceantigens of tumor cells. As set forth at col. 4, lines 45 et seq., thepatentees utilize autochthonous tumor cells in the development ofmonoclonal antibodies expressing active specific immunotherapy in humanneoplasia.

[0014] U.S. Pat. No. 5,693,763 teaches a glycoprotein antigencharacteristic of human carcinomas and not dependent upon the epithelialtissue of origin.

[0015] U.S. Pat. No. 5,783,186 is drawn to Anti-Her2 antibodies whichinduce apoptosis in Her2 expressing cells, hybridoma cell linesproducing the antibodies, methods of treating cancer using theantibodies and pharmaceutical compositions including said antibodies.

[0016] U.S. Pat. No. 5,849,876 describes new hybridoma cell lines forthe production of monoclonal antibodies to mucin antigens purified fromtumor and non-tumor tissue sources. U.S. Pat. No. 5,869,268 is drawn toa method for producing a human lymphocyte producing an antibody specificto a desired antigen, a method for producing a monoclonal antibody, aswell as monoclonal antibodies produced by the method. The patent isparticularly drawn to the production of an anti-HD human monoclonalantibody useful for the diagnosis and treatment of cancers.

[0017] U.S. Pat. No. 5,869,045 relates to antibodies, antibodyfragments, antibody conjugates and single chain immunotoxins reactivewith human carcinoma cells. The mechanism by which these antibodiesfunction is two-fold, in that the molecules are reactive with cellmembrane antigens present on the surface of human carcinomas, andfurther in that the antibodies have the ability to internalize withinthe carcinoma cells, subsequent to binding, making them especiallyuseful for forming antibody-drug and antibody-toxin conjugates. In theirunmodified form the antibodies also manifest cytotoxic properties atspecific concentrations.

[0018] U.S. Pat. No. 5,780,033 discloses the use of autoantibodies fortumor therapy and prophylaxis. However, this antibody is an antinuclearautoantibody from an aged mammal. In this case, the autoantibody is saidto be one type of natural antibody found in the immune system. Becausethe autoantibody comes from “an aged mammal”, there is no requirementthat the autoantibody actually comes from the patient being treated. Inaddition the patent discloses natural and monoclonal antinuclearautoantibody from an aged mammal, and a hybridoma cell line producing amonoclonal antinuclear autoantibody.

SUMMARY OF THE INVENTION

[0019] This application teaches a method for producing patient specificanti-cancer antibodies using a novel paradigm of screening. Theseantibodies can be made specifically for one tumor and thus make possiblethe customization of cancer therapy. Within the context of thisapplication, anti-cancer antibodies having either cell-killing(cytotoxic) or cell-growth inhibiting (cytostatic) properties willhereafter be referred to as cytotoxic. These antibodies can be used inaid of staging and diagnosis of a cancer, and can be used to treat tumormetastases.

[0020] The prospect of individualized anti-cancer treatment will bringabout a change in the way a patient is managed. A likely clinicalscenario is that a tumor sample is obtained at the time of presentation,and banked. From this sample, the tumor can be typed from a panel ofpre-existing anti-cancer antibodies. The patient will be conventionallystaged but the available antibodies can be of use in further staging thepatient. The patient can be treated immediately with the existingantibodies, and a panel of antibodies specific to the tumor can beproduced either using the methods outlined herein or through the use ofphage display libraries in conjunction with the screening methods hereindisclosed. All the antibodies generated will be added to the library ofanti-cancer antibodies since there is a possibility that other tumorscan bear some of the same epitopes as the one that is being treated.

[0021] In addition to anti-cancer antibodies, the patient can elect toreceive the currently recommended therapies as part of a multi-modalregimen of treatment. The fact that the antibodies isolated via thepresent methodology are relatively non-toxic to non-cancerous cellsallow combinations of antibodies at high doses to be used, either alone,or in conjunction with conventional therapy. The high therapeutic indexwill also permit re-treatment on a short time scale that should decreasethe likelihood of emergence of treatment resistant cells.

[0022] If the patient is refractory to the initial course of therapy ormetastases develop, the process of generating specific antibodies to thetumor can be repeated for re-treatment. Furthermore, the anti-cancerantibodies can be conjugated to red blood cells obtained from thatpatient and re-infused for treatment of metastases. There have been feweffective treatments for metastatic cancer and metastases usuallyportend a poor outcome resulting in death. However, metastatic cancersare usually well vascularized and the delivery of anti-cancer antibodiesby red blood cells can have the effect of concentrating the antibodiesat the site of the tumor. Even prior to metastases, most cancer cellsare dependent on the host's blood supply for their survival andanti-cancer antibody conjugated red blood cells can be effective againstin situ tumors, too. Alternatively, the antibodies may be conjugated toother hematogenous cells, e.g. lymphocytes, macrophages, monocytes,natural killer cells, etc.

[0023] There are five classes of antibodies and each is associated witha function that is conferred by its heavy chain. It is generally thoughtthat cancer cell killing by naked antibodies are mediated either throughantibody dependent cellular cytotoxicity or complement dependentcytotoxicity. For example murine IgM and IgG2a antibodies can activatehuman complement by binding the C-l component of the complement systemthereby activating the classical pathway of complement activation whichcan lead to tumor lysis. For human antibodies the most effectivecomplement activating antibodies are generally IgM and IgGl. Murineantibodies of the IgG2a and IgG3 isotype are effective at recruitingcytotoxic cells that have Fc receptors which will lead to cell killingby monocytes, macrophages, granulocytes and certain lymphocytes. Humanantibodies of both the IgGl and IgG3 isotype mediate ADCC.

[0024] Another possible mechanism of antibody mediated cancer killingmay be through the use of antibodies that function to catalyze thehydrolysis of various chemical bonds in the cell membrane and itsassociated glycoproteins or glycolipids, so-called catalytic antibodies.

[0025] There are two additional mechanisms of antibody mediated cancercell killing which are more widely accepted. The first is the use ofantibodies as a vaccine to induce the body to produce an immune responseagainst the putative cancer antigen that resides on the tumor cell. Thesecond is the use of antibodies to target growth receptors and interferewith their function or to down regulate that receptor so thateffectively its function is lost.

[0026] Accordingly, it is an objective of the invention to teach amethod for producing anti-cancer antibodies from cells derived from aparticular individual which are cytotoxic with respect to cancer cellswhile simultaneously being relatively non-toxic to non-cancerous cells.

[0027] It is an additional objective of the invention to produce novelanti-cancer antibodies.

[0028] It is a further objective of the instant invention to produceanti-cancer antibodies whose cytotoxicity is mediated through antibodydependent cellular toxicity.

[0029] It is yet an additional objective of the instant invention toproduce anti-cancer antibodies whose cytotoxicity is mediated throughcomplement dependent cellular toxicity.

[0030] It is still a further objective of the instant invention toproduce anti-cancer antibodies whose cytotoxicity is a function of theirability to catalyze hydrolysis of cellular chemical bonds.

[0031] Still an additional objective of the instant invention is toproduce anti-cancer antibodies useful as a vaccine to produce an immuneresponse against putative cancer antigen residing on tumor cells.

[0032] A further objective of the instant invention is the use ofantibodies to target cell membrane proteins, such as growth receptors,cell membrane pumps and cell anchoring proteins, thereby interferingwith or down regulating their function.

[0033] Yet an additional objective of the instant invention is theproduction of anti-cancer antibodies whose cell-killing utility isconcomitant with their ability to effect a conformational change incellular proteins such that a signal will be transduced to initiatecell-killing.

[0034] A still further objective of the instant invention is to produceanti-cancer antibodies which are useful for diagnosis, prognosis, andmonitoring of cancer, e.g. production of a panel of therapeuticanti-cancer antibodies to test patient samples to determine if theycontain any suitable antibodies for therapeutic use.

[0035] Yet another objective of the instant invention is to producenovel antigens, associated with cancer processes, which can bediscovered by using anti-cancer antibodies derived by the process of theinstant invention. These antigens are not limited to proteins, as isgenerally the case with genomic data; they may also be derived fromcarbohydrates or lipids or combinations thereof.

[0036] Other objects and advantages of this invention will becomeapparent from the following description wherein are set forth, by way ofillustration and example, certain embodiments of this invention.

DETAILED DESCRIPTION OF THE INVENTION

[0037] It is to be understood that while a certain form of the inventionis illustrated, it is not to be limited to the specific form orarrangement herein described and shown. It will be apparent to thoseskilled in the art that various changes may be made without departingfrom the scope of the invention and the invention is not to beconsidered limited to what is shown and described in the specification.

[0038] One of the potential benefits of monoclonal antibodies withrespect to the treatment of cancer is their ability to specificallyrecognize single antigens. It was thought that in some instances cancercells possess antigens that were specific to that kind of transformedcell. It is now more frequently believed that cancer cells have fewunique antigens, rather, they tend to over-express a normal antigen orexpress fetal antigens. Nevertheless, the use of monoclonal antibodiesprovided a method of delivering reproducible doses of antibodies to thepatient with the expectation of better response rates than withpolyclonal antibodies.

[0039] Traditionally, monoclonal antibodies have been made according tofundamental principles laid down by Kohler and Milstein. Mice areimmunized with antigens, with or without, adjuvants. The splenocytes areharvested from the spleen for fusion with immortalized hybridomapartners. These are seeded into microtitre plates where they can secreteantibodies into the supernatant that is used for cell culture. To selectfrom the hybridomas that have been plated for the ones that produceantibodies of interest the hybridoma supernatants are usually tested forantibody binding to antigens in an ELISA (enzyme linked immunosorbentassay) assay. The idea is that the wells that contain the hybridoma ofinterest will contain antibodies that will bind most avidly to the testantigen, usually the immunizing antigen. These wells are then subclonedin limiting dilution fashion to produce monoclonal hybridomas. Theselection for the clones of interest is repeated using an ELISA assay totest for antibody binding. Therefore, the principle that has beenpropagated is that in the production of monoclonal antibodies thehybridomas that produce the most avidly binding antibodies are the onesthat are selected from among all the hybridomas that were initiallyproduced. That is to say, the preferred antibody is the one with highestaffinity for the antigen of interest.

[0040] There have been many modifications of this procedure such asusing whole cells for immunization. In this method, instead of usingpurified antigens, entire cells are used for immunization. Anothermodification is the use of cellular ELISA for screening. In this methodinstead of using purified antigens as the target in the ELISA, fixedcells are used. In addition to ELISA tests, complement mediatedcytotoxicity assays have also been used in the screening process.However, antibody-binding assays were used in conjunction withcytotoxicity tests. Thus, despite many modifications, the process ofproducing monoclonal antibodies relies on antibody binding to the testantigen as an endpoint.

[0041] Most antibodies directed against cancer cells have been producedusing the traditional methods outlined above. These antibodies have beenused both therapeutically and diagnostically. In general, for both theseapplications, the antibody has been used as the targeting agent thatdelivers a payload to the site of the cancer. These antibody conjugatescan either be radioactive, toxic, or serve as an intermediary forfurther delivery of a drug to the body, such as an enzyme or biotin.Furthermore, it was widely held, until recently, that naked antibodieshad little effect in vivo. Both HERCEPTIN and RITUXIMAB are humanizedmurine monoclonal antibodies that have recently been approved for humanuse by the FDA. However, both these antibodies were initially made byassaying for antibody binding and their direct cytotoxicity was not theprimary goal during the production of hybridomas. Any tendency for theseantibodies to produce tumor cell killing is thus through chance, not bydesign.

[0042] Although the production of monoclonal antibodies have beencarried out using whole cell immunization for various applications thescreening of these hybridomas have relied on either putative oridentified target antigens or on the selectivity of these hybridomas forspecific tissues. It is axiomatic that the best antibodies are the oneswith the highest binding constants. This concept originated from thebasic biochemical principle that enzymes with the highest bindingconstants were the ones that were the most effective for catalyzing areaction. This concept is applicable to receptor ligand binding wherethe drug molecule binding to the receptor with the greatest affinityusually has the highest probability for initiating or inhibiting asignal. However, this may not always be the case since it is possiblethat in certain situations there may be cases where the initiation orinhibition of a signal may be mediated through non-receptor binding. Theinformation conveyed by a conformational change induced by ligandbinding can have many consequences such as a signal transduction,endocytosis, among the others. The ability to produce a conformationalchange in a receptor molecule may not necessarily be due to the fillingof a ligand receptor pocket but may occur through the binding of anotherextra cellular domain or due to receptor clustering induced by amultivalent ligand.

[0043] The production of antibodies to produce cell killing need not bepredicated upon screening of the hybridomas for the best bindingantibodies. Rather, although not advocated by those who producemonoclonal antibodies, the screening of the hybridoma supernatants forcell killing or alternatively for cessation of growth of the cancerouscells may be selected as a desirable endpoint for the production ofcytotoxic or cytostatic antibodies. It is well understood that thein-vivo antibodies mediate their function through the Fc portions andthat the utility of the therapeutic antibody is determined by thefunctionality of the constant region or attached moieties. In this casethe FAb portion of the antibody, the antigen-combining portion, willconfer to the antibody its specificity and the Fc portion itsfunctionality. The antigen combining site of the antibody can beconsidered to be the product of a natural combinatorial library. Theresult of the rearrangement of the variable region of the antibody canbe considered a molecular combinatorial library where the output is apeptide. Therefore, the sampling of this combinatorial library can bebased on any parameter. Like sampling a natural compound library forantibiotics, it is possible to sample an antibody library for cytotoxicor cytostatic compounds.

[0044] The various endpoints in a screen must be differentiated fromeach other. For example, the difference between antibody binding to thecell is distinct from cell killing. Cell killing (cytotoxicity) isdistinct from the mechanisms of cell death such as oncosis or apoptosis.There can be many processes by which cell death is achieved and some ofthese can lead either to oncosis or apoptosis. There is speculation thatthere are other cell death mechanisms other than oncosis or apoptosisbut regardless of how the cell arrives at death there are somecommonalities of cell death. One of these is the absence of metabolismand another is the denaturation of enzymes. In either case vital stainswill fail to stain these cells. These endpoints of cell death have beenlong understood and predate the current understanding of the mechanismsof cell death. Furthermore, there is the distinction between cytotoxiceffects where cells are killed and cytostatic effects where theproliferation of cells are inhibited.

[0045] In a preferred embodiment of the present invention, the assay isconducted by focusing on cytotoxic activity toward cancerous cells as anend point. In a preferred embodiment, a live /dead assay kit , forexample the LIVE/DEAD® Viability/Cytotoxicity Assay Kit (L-3224) byMolecular Probes, is utilized. The Molecular Probes kit provides atwo-color fluorescence cell viability assay that is based on thesimultaneous determination of live and dead cells with two probes thatmeasure two recognized parameters of cell viability intracellularesterase activity and plasma membrane integrity. The assay principlesare general and applicable to most eukaryotic cell types, includingadherent cells and certain tissues, but not to bacteria or yeast. Thisfluorescence-based method of assessing cell viability is preferred inplace of such assays as trypan blue exclusion, is Cr release and similarmethods for determining cell viability and cytotoxicity.

[0046] In carrying out the assay, live cells are distinguished by thepresence of ubiquitous intracellular esterase activity, determined bythe enzymatic conversion of the virtually nonfluorescent cell-permeantCALCEIN AM to the intensely fluorescent Calcein. The polyanionic dyeCalcein is well retained within live cells, producing an intense uniformgreen fluorescence in live cells (ex/em ˜495 nm/˜515 nm). EthD-1 enterscells with damaged membranes and undergoes a 40-fold enhancement offluorescence upon binding to nucleic acids, thereby producing a brightred fluorescence in dead cells (ex/em ˜495 nm/˜635 nm). EthD-1 isexcluded by the intact plasma membrane of live cells. The determinationof cell viability depends on these physical and biochemical propertiesof cells. Cytotoxic events that do not affect these cell properties maynot be accurately assessed using this method. Background fluorescencelevels are inherently low with this assay technique because the dyes arevirtually nonfluorescent before interacting with cells.

[0047] In addition to the various endpoints for screening, there are twoother major characteristics of the screening process. The library ofantibody gene products is not a random library but is the product of abiasing procedure. In the examples below, the biasing is produced byimmunizing mice with fixed cells. This increases the proportion ofantibodies that have the potential to bind the target antigen. Althoughimmunization is thought of as a way to produce higher affinityantibodies (affinity maturation) in this case it is not. Rather, it canbe considered as a way to shift the set of antigen combining sitestowards the targets. This is also distinct from the concept of isotypeswitching where the functionality, as dictated by the constant portionof the heavy chain, is altered from the initial IgM isotype to anotherisotype such as IgG.

[0048] The third key feature that is crucial in the screening process isthe use of multitarget screening. To a certain extent specificity isrelated to affinity. An example of this is the situation where anantigen has very limited tissue distribution and the affinity of theantibody is a key determinant of the specificity of the antibody-thehigher the affinity the more tissue specific the antibody and likewisean antibody with low affinity may bind to tissues other than the one ofinterest. Therefore, to address the specificity issue the antibodies arescreened simultaneously against a variety of cells. In the examplesbelow the hybridoma supernatants (representing the earliest stages ofmonoclonal antibody development), are tested against a number of celllines to establish specificity as well as activity.

[0049] The antibodies are designed for therapeutic treatment of cancerin patients. Ideally the antibodies can be naked antibodies. They canalso be conjugated to toxins. They can be used to target other moleculesto the cancer. e.g. biotin conjugated enzymes. Radioactive compounds canalso be used for conjugation.

[0050] The antibodies can be fragmented and rearranged molecularly. Forexample Fv fragments can be made; sFv-single chain Fv fragments;diabodies etc.

[0051] It is envisioned that these antibodies can be used for diagnosis,prognosis, and monitoring of cancer. For example the patients can haveblood samples drawn for shed tumor antigens which can be detected bythese antibodies in different formats such as ELISA assays, rapid testpanel formats etc. The antibodies can be used to stain tumor biopsiesfor the purposes of diagnosis. In addition a panel of therapeuticantibodies can be used to test patient samples to determine if there areany suitable antibodies for therapeutic use.

EXAMPLE ONE

[0052] In order to produce monoclonal antibodies specific for a tumorsample the method of selection of the appropriate hybridoma wells iscomplicated by the probability of selecting wells which will producefalse positive signals. That is to say that there is the likelihood ofproducing antibodies that can react against normal cells as well ascancer cells. To obviate this possibility one strategy is to mask theanti-normal antigen antibodies from the selection process. This can beaccomplished by removing the anti-normal antibodies at the first stageof screening thereby revealing the presence of the desired antibodies.Subsequent limiting dilution cloning can delineate the clones that willnot produce killing of control cells but will produce target cancer cellkilling.

[0053] Biopsy specimens of breast, melanoma, and lung tumors wereobtained and stored at −70° C. until used. Single cell suspensions wereprepared and fixed with −30° C., 70% ethanol, washed with PBS andreconstituted to an appropriate volume for injection. Balb/c mice wereimmunized with 2.5×10⁵−1×10⁶ cells and boosted every third week until afinal pre-fusion boost was performed three days prior to thesplenectomy. The hybridomas were prepared by fusing the isolatedsplenocytes with Sp2/0 and NS1 myeloma partners. The supernatants fromthe fusions were tested for subcloning of the hybridomas. Cells(including A2058 melanoma cells, CCD-12CoN fibroblasts, MCF-12A breastcells among others) were obtained from ATCC and cultured according toenclosed instructions. The HEY cell line was a gift from Dr. InkaBrockhausen. The non-cancer cells, e.g. CCD-12CoN fibroblasts andMCF-12A breast cells, were plated into 96-well microtitre plates (NUNC)1 to 2 weeks prior to screening. The cancer cells, e.g. HEY, A2058, BT483, and HS294t, were plated two or three days prior to screening.

[0054] The plated normal cells were fixed prior to use. The plates werewashed with 100 microliters of PBS for 10 minutes at room temperatureand then aspirated dry. 75 microliters of 0.01 percent glutaraldehydediluted in PBS were added to each well for five minutes and thenaspirated. The plates were washed with 100 microliters of PBS threetimes at room temperature. The wells were emptied and 100 microliters ofone percent human serum albumin in PBS was added to each well for onehour at room temperature. The plates were then stored at four degreesCelsius.

[0055] Prior to the transfer of the supernatant from the hybridomaplates the fixed normal cells were washed three times with 100microliters of PBS at room temperature. After aspiration to themicroliters of the primary hybridoma culture supernatants weretransferred to the fixed cell plates and incubated for two hours at 37degrees Celsius in a 8 percent C0₂ incubator. The hybridoma supernatantsderived from melanoma was incubated with CCD-12 CoN cells and thosederived from breast cancer were incubated with MCF-12a cells. Afterincubation the absorbed supernatant was divided into two 75 microliterportions and transferred to target cancer cell plates. Prior to thetransfer the cancer cell plates were washed three times with 100microliters of PBS. The supernatant from the CCD-12 CoN cells weretransferred to the A2058 and the HS294t cells, whereas the supernatantfrom MCF-12A cells were transferred to the HEY and BT 483 cells. Thecancer cells were incubated with the hybridoma supernatants for 18 hoursat 37 degrees Celsisu in an 8 percent CO₂ incubator.

[0056] The Live/Dead cytotoxicity assay was obtained from MolecularProbes (Eu,Oreg.). The assays were performed according to themanufacturer's instructions with the changes outlined below. The plateswith the cells were washed once with 100 microliters of PBS at 37° C. 75to 100 microliters of supernatant from the hybridoma microtitre plateswere transferred to the cell plates and incubated in a 8% Co₂ incubatorfor 18-24 hours. Then, the wells that served as the all dead controlwere aspirated until empty and 50 microliters of 70% ethanol was added.The plate was then emptied by inverting and blotted dry. Roomtemperature PBS was dispensed into each well from a multichannel squeezebottle, tapped three times, emptied by inversion and then blotted dry.50 microliters of the fluorescent Live/Dead dye diluted in PBS was addedto each well and incubated at 37° C. in a 5% CO₂ incubator for one hour.The plates were read in a Perkin-Elmer HTS7000 fluorescence plate readerand the data was analyzed in Microsoft Excel.

[0057] Four rounds of screening were conducted to produce single clonehybridoma cultures. For two rounds of screening the hybridomasupernatants were tested only against the cancer cells. In the lastround of screening the supernatant was tested against a number ofnon-cancer cells as well as the target cells indicated in table 1. Theantibodies were isotyped using a commercial isotyping kit.

[0058] A number of monoclonal antibodies were produced in accordancewith the method of the present invention. These antibodies, whosecharacteristics are summarized in Table 1, are identified as 3BD-3,3BD-6, 3BD-8, 3BD-9, 3BD-15, 3BD-25, 3BD-26 and 3BD-27. Each of thedesignated antibodies is produced by a hybridoma cell line depositedwith the American Type Culture Collection at 10801 University Boulevard,Manassas, Va. having an ATCC Accession Number as follows: Antibody ATCCAccession Number 3BD-3 3BD-6 3BD-8 3BD-9 3BD-15 3BD-25 3BD-26 3BD-27

[0059] These antibodies are considered monoclonal after four rounds oflimiting dilution cloning. The anti-melanoma antibodies did not producesignificant cancer cell killing. The panel of anti-breast cancerantibodies killed 32-87% of the target cells and <1-3% of the controlcells. The predominant isotype was IgGl even though it was expected thatthe majority of anti-tumor antibodies would be directed againstcarbohydrate antigens, and thus, be of the IgM type. There is a hightherapeutic index since most antibodies spare the control cells fromcell death. TABLE 1 Anti-Breast Cancer Antibodies % Cell Death Targetfor Anti-Breast Normal Fibroblast Cancer Antibodies Cells FibrocysticBreast Cells Clones (HEY & A2058) (CCD-12CoN) (NCF-12A) Isotype 3BD-374.9% 3.7%  <1% y1, λ 3BD-6 68.5% 5.6%  <1% y1, λ 3BD-8 81.9% 4.5% 2.6%y1, η 3BD-9 77.2% 7.9%  1% y1, λ 3BD-15 87.1%  <1%  <1% y1, λ 3BD-2654.8% 3.3%  <1% μ, η 2BD-25 32.4% 3.6%  <1% y1, η 3BD-27 60.1% 8.3% 1.3%y1, η

EXAMPLE 2

[0060] In this example customized anti-cancer antibodies are produced byfirst obtaining samples of the patient's tumor. Usually this is from abiopsy specimen from a solid tumor or a blood sample from hematogenoustumors. The samples are prepared into single cell suspensions and fixedfor injection into mice. After the completion of the immunizationschedule the hybridomas are produced from the splenocytes. Thehybridomas are screened against a variety of cancer cell lines andnormal cells in standard cytotoxicity assays. Those hybridomas that arereactive against cancer cell lines but are not reactive against normalnon-transformed cells are selected for further propagation. Clones thatwere considered positive were ones that selectively killed the cancercells but did not kill the non-transformed cells. The antibodies arecharacterized for a large number of biochemical parameters and thenhumanized for therapeutic use.

[0061] The melanoma tumor cells isolated and cell lines were cultured asdescribed in Example 1. Balb/c mice were immunized according to thefollowing schedule: 200,000 cells s.c. and i.p. on day 0, then 200,000cells were injected i.p. on day 21, then 1,000,000 cells were injectedon day 49, then 1,250,000 cells in Freund's Complete Adjuvant wereinjected i.p. on day 107, and then 200,000 cells were injected on day120 i.p. and then the mice were sacrificed on day 123. The spleens wereharvested and the splenocytes were divided into two aliquots for fusionwith Sp2/0 (1LN) or NS-1 (2LN) myeloma partners using the methodsoutlined in example 1.

[0062] The screening was carried out 11 days after the fusion againstA2058 melanoma cells and CCD-12CoN fibroblasts. Each pair of plates werewashed with 100 microliters of room temperature PBS and then aspiratedto near dryness. Then 50 microliters of hybridoma supernatant was addedto the same wells on each of the two plates. The spent Sp2/0 supernatantwas added to the control wells at the same volume and the plates wereincubated for around 18 hours at 37 degrees Celsius at a 8%CO₂, 98%relative humidity incubator. Then each pair of plates were removed andin the positive control wells 50 microliters of 70% ethanol wassubstituted for the media for 4 seconds. The plates were then invertedand washed with room temperature PBS once and dried. Then 50 uL offluorescent live/dead dye diluted in PBS (Molecular Probes Live/DeadKit) was added for one hour and incubated at 37 degrees Celsius. Theplates were then read in a Perkin-Elmer fluorescent plate reader and thedata analyzed using Microsoft Excel. The wells that were consideredpositive were subcloned and the same screening process was repeated 13days later and then 33 days later. The results of the last screening isoutlined in Table 2 below. A number of monoclonal antibodies wereproduced in accordance with the method of the present invention. Theseantibodies, whose characteristics are summarized in Table 2, areidentified as 1LN-1, 1LN-8, 1LN-12, 1LN-14, 2LN-21, 2LN-28, 2LN-29,2LN-31, 2LN-33, 2LN-34 and 2LN-35. Each of the designated antibodies isproduced by a hybridoma cell line deposited with the American TypeCulture Collection at 10801 University Boulevard, Manassas, Va. havingan ATCC Accession Number as follows: Antibody ATCC Accession Number1LN-1 1LN-8 1LN-12 1LN-14 2LN-21 2LN-28 2LN-29 2LN-31 2LN-33 2LN-342LN-35

[0063] TABLE 2 Anti-Melanoma Antibodies % Cell Death Target for Anti-Melanoma Normal Fibroblast Antibodies Cells Clones (A2058) (CCD-1 2CoN)1LN-1  59.4%   <1% 1LN-8  11.0%  5.0% 1LN-12  55.2%  1.4% 1LN-14  51.4%  <1% 2LN-21  72.0% 15.9% 2LN-28  66.6% 12.4% 2LN-29  78.2%  6.1% 2LN-31100%  7.8% 2LN-33  94.2%   <1% 2LN-34  56.6% 11.2% 2LN-35  66.5%  5.6%

[0064] The table illustrates that clones from both the Sp2/0 and NS-lfusions were able to produce antibodies that had a greater than 50%killing rate for cancerous cells and at the same time some of the cloneswere able to produce less than one percent killing of normal controlfibroblasts.

EXAMPLE 3

[0065] In this example antibodies were produced to several differentbreast tumor samples following the method of Example 2 in order todemonstrate the generality of producing customized antibodies. Biopsyspecimens of breast tumors were obtained and stored at −70° C until usedas noted in Example 1. Single cell suspensions were prepared for eachspecimen and fixed with −30° C., 70% ethanol, washed with PBS andreconstituted to an appropriate volume for injection. Female, 7-8 weekold, A strain, H-2^(d) haplotype Balb/c mice (Charles River Canada, St.Constant, QC, Can), were immunized with 2.5×10⁵−1×10⁶ cells and boostedevery third week until a final pre-fusion boost was performed three daysprior to the splenectomy. The hybridomas were prepared by fusing theisolated splenocytes with Sp2/0 myeloma partners. The supernatants fromthe fusions were tested for subcloning of the hybridomas.

[0066] Hs574.T breast ductal carcinoma cells, A2058 melanoma cells,NCI-H460 human lung large cell carcinoma, NCI-H661 human lung large cellcarcinoma, CCD-112CoN human colon fibroblasts, CCD-27sk human skinfibroblasts, MCF-12A human mammary epithelial cells, Hs574.mg humanbreast cells and other cell lines, were obtained from ATCC and culturedaccording to enclosed instructions. Both cancer and non-cancer cellswere plated three to four days prior to screening.

[0067] The hybridomas were cultured for ten to twelve days after fusionand observed under the microscope. When 20 to 25% of the wells were over80% confluent, the hybridoma supernatants were screened in acytotoxicity assay. The hybridoma supernatants were divided into two 75microliter portions; one portion was added to a target cancer cell plateand another to a non-cancer cell plate. Prior to transfer of hybridomasupernatants, the cell plates were washed three times with 100microliters of PBS. The supernatant from the anti-breast cancerhybridomas were transferred to the Hs574.T and the Hs574.mg cells,whereas the supernatant from the anti-lung cancer hybridoma weretransferred to the NCI-H460 and CCD-27SK cells. The cancer cells wereincubated with the hybridoma supernatants for 18 hours at 37 degreesCelsius in an 8 percent CO₂ incubator.

[0068] The Live/Dead cytotoxicity assay was obtained from MolecularProbes (Eugene, Oreg.). The assays were performed according to themanufacturer's instructions with the changes outlined below. The plateswith the cells were washed once with 100 microliters of PBS at 37° C. 75to 100 microliters of supernatant from the hybridoma microtitre plateswere transferred to the cell plates and incubated in a 8% CO₂ incubatorfor 18-24 hours. Then, the wells that served as the dead control cellswere aspirated until empty and 50 microliters of 70% ethanol was added.The plate was then emptied by inverting and blotted dry. Roomtemperature PBS was dispensed into each well from a multichannel squeezebottle, tapped three times, emptied by inversion and then blotted dry.50 microliters of the fluorescent Live/Dead dye diluted in PBS was addedto each well and incubated at 37° C. in a 5% CO₂ incubator for one hour.The plates were read in a Perkin-Elmer HTS7000 fluorescence plate readerand the data was analyzed in Microsoft Excel (Microsoft, Redmond,Wash.).

[0069] Four rounds of screening were conducted to produce single clonehybridoma cultures. For two rounds of screening the hybridomasupernatants were tested only against the cancer cells. In the lastround of screening the supernatant was tested against a number ofnon-cancer cells as well as the target cells indicated in Table 3. Theantibodies were isotyped using a commercial isotyping kit (Roche,Indianapolis, Ind.).

[0070] A number of monoclonal antibodies were produced in accordancewith the method of the present invention. These antibodies, whosecharacteristics are summarized in Table 3, are identified as 4BD-1,4BD-3, 4BD-6, 4BD-9, 4BD-13, 4BD-18, 4BD-20, 4BD-25, 4BD-37, 4BD-32,4BD-26, 4BD-27, 4BD-28, 4BD-50, 6BD-1, 6BD-3, 6BD-5, 6BD-11, 6BD-25,7BD-7, 7BD-12-1, 7BD-12-2, 7BD-13, 7BD-14, 7BD-19, 7BD-21, 7BD-24,7BD-29, 7BD-30, 7BD-31, 7BDI-17, 7BDI-58, 7BDI-60 and 7BDI-62. Each ofthe designated antibodies is produced by a hybridoma cell line depositedwith the American Type Culture Collection at 10801 University Boulevard,Manassas, Va. having an ATCC Antibody ATCC Accession Number 4BD-1 4BD-34BD-6 4BD-9 4BD-13 4BD-18 4BD-20 4BD-25 4BD-37 4BD-32 4BD-26 4BD-274BD-28 4BD-50 6BD-1 6BD-3 6BD-5 6BD-11 6BD-25 7BD-7 7BD-12-1 7BD-12-27BD-13 7BD-14 7BD-19 7BD-21 7BD-24 7BD-29 7BD-30 7BD-31 7BDI-17 7BDI-587BDI-60 7BDI-62

[0071] These antibodies are considered monoclonal after four rounds oflimiting dilution cloning. The panel of anti-breast cancer antibodieskilled 15-79% of the target cells and <1-31% of the control cells. Themajority of anti-tumor antibodies were IgM type, suggesting they couldbe directed against carbohydrate antigens on the surface of tumor cells.There is a high therapeutic index since most of the antibodies do notcause the normal cells to undergo cell death.

[0072] These monoclonal antibodies are characterized for a number ofimmunological and biochemical parameters. A cell based enzyme linkedimmunosorbent assay (ELISA) was established for measuring the binding ofthe antibodies derived of each clones to different cell lines. Cellswere seeded and grown on 96-well tissue culture plates. The plates werewashed with 100 microliters of PBS. 100 microliters of cold 4 percentparaformaldehyde in PBS were added to each well for ten minutes and thenaspirated. The plates were washed with PBS using a multichannel squeezebottle . The wells were emptied and 100 microliters of blocking buffer(1 percent hydrocasein, 0.1 percent geletin in 50 mM Tris-HCl buffer, pH9.3) was added to each well for one hour at room temperature. The plateswere washed three times with a buffer (0.05 percent Tween 20 in 10 mMPBS) at room temperature and then stored at −30 degrees Celsius with 100microliters of the McHale & Slavin, P.A. 2056.009 36 buffer. Prior touse the plates were thawed and the buffer was aspirated from each well.75 microliters of hybridoma supernatant were added to each well andincubated for 60 minutes at room temperature. After the plates werewashed with PBS using a multichannel squeeze bottle, 50 microliters of acombination of peroxidase conjugated goat anti-mouse IgG and peroxidaseconjugated donkey anti-mouse IgM (Jackson ImmunoResearch Lab, Inc., WestGrove, Pa.) was added and incubated for 30 minutes at room temperature.After the last wash, 50 microliters of orthophenylene diamine (OPD)(Sigma, St. Louis, Mo.) was added to each well and the optical densitywas read at 492 nm on the HTS7000 plate reader after adding equal volumeof 1 N sulfuric acid. Different clones show different profiles inbinding to different cells (Table 3). This indicates that they maytarget different cell surface antigen and further suggests the variabledistribution of these antigen on the surface of cancer cells. Thosebinding only to cancer cells but not to normal cells could identifycertain tumor-associated antigen. TABLE 3 Anti-Breast Cancer Antibodies% Cell Death Binding to cell lines Clones Isotype Hs574.T Hs574.mgHs574.T Hs574.mg NCl-H460 CCD-27sk A2058 6BD-1 μ, κ 38.2 5 0.8 0.5 0.60.3 ND* 6BD-3 μ, κ 79 12 0.35 0.25 0.24 0.14 ND 6BD-5 μ, κ 57.3 8 1.00.3 0.14 0.25 ND 6BD-11 μ, κ 52.3 11 0.15 0.1 0.17 0.1 ND 6BD-25 μ, κ33.3 2 0.15 0.1 0.2 0.1 ND 4BD-26 μ, κ 27 1.8 0.5 ND ND <0.1 ND 4BD-27μ, κ 19.6 <1 0.9 ND ND 0.5 ND 4BD-28 μ, κ 26.4 <1 0.8 ND ND <0.1 ND4BD-32 μ, κ 41.7 4 0.8 ND ND <0.1 ND 4BD-50 μ, κ 20 <1 0.8 ND ND <0.1 ND4BD-1 μ, κ 23 31 0.6 ND ND <0.1 ND 4BD-3 μ, κ 29.7 8.2 1.7 ND ND 0.1 ND4BD-6 μ, κ 17 <1 0.9 ND ND <0.1 ND 4BD-9 μ, κ 15 <1 0.6 ND ND <0.1 ND4BD-13 μ, κ 31 <1 1.2 ND ND <0.1 ND 4BD-18 μ, κ 23.3 2.4 0.7 ND ND 0.12ND 4BD-20 μ, κ 45 <1 0.95 ND ND <0.1 ND 4BD-25 μ, κ 26 14.16 1.8 ND ND0.1 ND 4BD-37 μ, κ 30 <1 0.8 ND ND <0.1 ND 7BD-7 μ, κ 24 3 0.8 0.3 1.40.26 ND 7BD-12-1 μ, κ 22 6 0.36 0.16 0.43 0.1 ND 7BD-12-2 μ, κ 31 2 0.20.2 0.2 0.2 0.2 7BD-13 μ, κ 29 12 0.1 0.15 0.2 0.1 0.2 7BD-14 μ, κ 32 130.4 0.4 0.6 0.3 0.5 7BD-19 μ, κ 20 4 1.3 0.4 0.43 0.2 ND 7BD-21 μ, κ 2113 0.4 0.5 0.25 0.3 ND 7BD-24 μ, κ 32 15 0.3 0.1 0.14 0.15 ND 7BD-29 1516 0.3 0.24 0.14 0.16 ND 7BD-30 μ, κ 23 13 0.34 0.24 0.2 0.16 ND 7BD-31μ, κ 28 10 0.3 0.4 0.4 0.3 0.4 7BDI-17 μ, κ 23 <1 0.75 ND ND ND ND7BDI-58 γ1, κ 17.5 <1 0.77 ND ND ND ND 7BDI-60 γ1, κ 15 <1 0.73 ND ND NDND 7BDI-62 15 5 0.55 ND ND ND ND

EXAMPLE 4

[0073] In this example customized anti-cancer antibodies are produced toa lung cancer sample by first obtaining samples of the patient's tumorpreparing single cell suspensions which are then fixed for injectioninto mice as noted in Example 1. After the completion of theimmunization schedule the hybridomas are produced from the splenocytes.The hybridomas are screened against a variety of cancer cell lines andnormal cells in standard cytotoxicity assays. Those hybridomas that arereactive against cancer cell lines but are not reactive against normalnon-transformed cells are selected for further propagation. Clones thatwere considered positive were ones that selectively killed the cancercells but did not kill the non-transformed cells.

[0074] The lung cancer cells were isolated and cell lines were culturedas described in Example 1. Female, 7-8 week old, A strain, H-2^(d)haplotype Balb/c mice (Charles River Canada, St. Constant, QC, Can),were immunized with human lung cancer cells. The lung cancer cellsuspensions were emulsified in an equal volume of Freund's completeadjuvant (FCA) for the first immunization and then in Freund'sincomplete adjuvant (FIA) for subsequent immunizations at 0, 21, 45days. 5×10⁵ cells were used to immunize each mouse either through asubcutaneous or intra-peritoneal route. Immunized mice were sacrificed3-4 days after the final immunization with human lung cancer cells at148 days, given-intra-peritoneally, in PBS at pH 7.4. The spleens wereharvested and the splenocytes were divided into two aliquots for fusionwith Sp2/0 myeloma partners using the methods outlined in Example 1.

[0075] The screening was carried out 10 days after the fusion againstNCI-H460 and/or NCI-H661 cells and CCD-27SK fibroblasts. Each pair ofplates were washed with 100 microliters of room temperature PBS and thenaspirated to near dryness. Then 75 microliters of hybridoma supernatantwas added per well on each of the two plates. The spent Sp2/0supernatant was added to the control wells at the same volume and theplates were incubated for around 18 hours at 37 degrees Celsius at a8%CO₂, 98% relative humidity incubator. Then each pair of plates wasremoved and in the positive control wells 50 microliters of 70% ethanolwas substituted for the media for 4 seconds. The plates were theninverted and washed with room temperature PBS once and dried. Then 50microliters of fluorescent live/dead dye diluted in PBS (MolecularProbes Live/Dead Kit) was added for one hour and incubated at 37 degreesCelsius. The plates were then read in a Perkin-Elmer fluorescent platereader and the data analyzed using Microsoft Excel. The wells that wereconsidered positive were subcloned and the same screening process wasrepeated 6 days later and then 13 days later. The result of the lastscreening is outlined in Table 4 below. Antibodies were characterizedfor binding to different cell lines with a cellular ELISA according tothe methods of Example 3. A number of monoclonal antibodies wereproduced in accordance with the method of the present invention. Theseantibodies, whose characteristics are summarized in Table 4, areidentified as 5LAC2, 5LAC4, 5LAC20, and 5LAC23. Each of the designatedantibodies is produced by a hybridoma cell line deposited with theAmerican Type Culture Collection at 10801 University Boulevard,Manassas, Va. having an ATCC Accession Number as follows: Antibody ATCCAccession Number 5LAC2 5LAC4 5LAC20 5LAC23.

[0076] TABLE 4 Anti-Lung Cancer Antibodies % Cell Death Binding to celllines Clones Isotype Hs574.T NCl-H460 NCl-H661 A2058 CCD-27sk Hs574.THs574.mg NCl-H460 CCD-27sk A2058 5LAC2 μ, κ 30 7 45.3 23 <1 0.2 0.2 0.260.2 0.2 5LAC4 μ, κ 21 11 20.5 23 3 0.7 0.9 1.7 0.8 0.9 5LAC20 μ, κ 23 766.4 24 3 0.5 0.2 0.6 0.2 0.2 23 8 57.6 25 5 0.6 0.6 0.6 0.6 0.6

[0077] The table illustrates that clones were able to produce antibodiesthat had a greater than 7-67% killing rate for cancerous cells and atthe same time some of the clones were able to produce less than fivepercent killing of normal control fibroblasts.

EXAMPLE 5

[0078] In this example customized anti-cancer antibodies are produced toa patient's lung cancer cells, but cultured cells were used in theantibody development process to demonstrate the generality of theimmunization process. The samples were prepared into single cellsuspensions and fixed for injection into mice as noted in Example 1.After the completion of three rounds of immunization with cells deriveddirectly from a patient's lung cancer, the mice were immunized twicewith a human lung large cell carcinoma cell line (NCI-H460). Hybridomaswere produced from splenocytes and the supernatants were screenedagainst a variety of cancer cell lines and normal cells in standardcytotoxicity assays. Those hybridomas that were reactive against cancercell lines but were not reactive against normal non-transformed cellswere selected for further propagation. Clones that were consideredpositive were ones that selectively killed the cancer cells but did notkill the non-transformed cells. The antibodies are characterized for alarge number of biochemical parameters and then humanized fortherapeutic use.

[0079] The lung tumor cells isolated and cell lines were cultured asdescribed in Example 1. Balb/c mice, A strain with H-2^(d) haplotypefrom Charles River Canada, St. Constant, Quebec, Canada, female, 7-8week old, were immunized with the human lung cancer cells emulsified inan equal volume of either Freund's complete adjuvant (FCA) for the firstimmunization and then in Freund's incomplete adjuvant (FIA) forsubsequent immunizations at 0, 21, 45 days with 5×10⁵ cells. The micewere immunized with fixed NCI H460 cells, which were prepared from NCIH460 cells grown in T-75 cell culture flask by scraping mono-layer cellsinto cell suspensions at 105, 150 and 170 days. Immunized mice weresacrificed 3-4 days after the final immunization with NCI H460 cells,given intra-peritoneally, in phosphate buffered saline buffer (PBS), pH7.4. The spleens were harvested and the splenocytes were divided intotwo aliquots for fusion with Sp2/0 myeloma partners using the methodsoutlined in Example 1.

[0080] The screening was carried out 10 days after the fusion againstNCI H460 cells and CCD-27SK fibroblasts as described in Example 4.Antibodies were characterized for binding to different cell lines with acellular ELISA according to the methods of Example 3.

[0081] The wells that were considered positive were subcloned and thesame screening process was repeated 9 days and 18 days later. Theresults are outlined in Table 5 below. A number of monoclonal antibodieswere produced in accordance with the method of the present invention.These antibodies, whose characteristics are summarized in Table 5, areidentified as H460-1, H460-4, H460-5, H460-10, H460-14, H460-16-1,H460-16-2, H460-23 and H460-27. Each of the designated antibodies isproduced by a hybridoma cell line deposited with the American TypeCulture Collection at 10801 University Boulevard, Manassas, Va. havingan ATCC Accession Number as follows: Antibody ATCC Accession NumberH460-1 H460-4 H460-5 H460-10 H460-14 H460-16-1 H460-16-2 H460-23 H460-27

[0082] TABLE 5 Anti-Lung Cancer Antibodies % Cell Death Binding to celllines Clones Isotype NCl-H460 Hs574.T A2058 CCD- Hs574. Hs574.m NCl-CCD- A2058 H460-1 16 30 23 <1 1.0 0.6 0.5 0.7 ND H460-4 37 21 23 3 1.00.6 0.4 0.6 ND H460-5 μ, κ 22.5 23 24 3 1.0 0.3 0.3 0.2 ND H460-10 μ, κ8 23 25 5 0.3  0.14 0.2 0.1 ND H460-14 17 ND ND 4 1.1 0.6 0.4 0.54 NDH460-16-1 33 ND ND 8 1.0 0.6 0.3 0.5 ND H460-16-2 22 ND ND 3 1.0 0.6 0.30.7 ND H460-22-1 21 ND ND 5 0.6 0.4 0.3 0.4 ND H460-22-2 μ, κ 23 ND ND 30.4 0.1 0.1 0.1 ND H460-23 μ, κ 36 36 18 1 0.4 1.1 0.54 0.53 0.58H460-27 μ, κ 33 31 16 8 0.3 0.4 0.4 0.3 0.4

[0083] The table illustrates that clones were able to produce antibodiesthat had a greater than 15% killing rate for cancerous cells and at thesame time some of the clones were able to produce less than eightpercent killing of normal control fibroblasts.

[0084] The anti-cancer antibodies of the invention are useful fortreating a patient with a cancerous disease when administered inadmixture with a pharmaceutically acceptable adjuvant, for examplenormal saline, a lipid emulsion, albumen, phosphate buffered saline orthe like and are administered in an amount effective to mediatetreatment of said cancerous disease, for example with a range of about 1microgram per milliliter to about 1 gram per milliliter.

[0085] The method for treating a patient suffering from a cancerousdisease may further include the use of conjugated anti-cancer antibodiesand would this include conjugating patient specific anti-cancerantibodies with a member selected from the group consisting of toxins,enzymes, radioactive compounds, and hematogenous cells; andadministering these conjugated antibodies to the patient; wherein saidanti-cancer antibodies are administered in admixture with apharmaceutically acceptable adjuvant, for example normal saline, a lipidemulsion, albumen, phosphate buffered saline or the like and areadministered in an amount effective to mediate treatment of saidcancerous disease, for example with a range of about 1 microgram per milto about 1 gram per mil. In a particular embodiment, the anti-cancerantibodies useful in either of the above outlined methods may be ahumanized antibody.

[0086] The anti-cancer antibodies of the invention are useful fortreating a patient with a cancerous disease when administered inadmixture with a pharmaceutically acceptable adjuvant, for examplenormal saline, a lipid emulsion, albumen, phosphate buffered saline orthe like and are administered in an amount effective to mediatetreatment of said cancerous disease, for example with a range of about 1microgram per mil to about 1 gram per mil.

[0087] The method for treating a patient suffering from a cancerousdisease may further include the use of conjugated anti-cancer antibodiesand would this include conjugating patient specific anti-cancerantibodies with a member selected from the group consisting of toxins,enzymes, radioactive compounds, and hematogenous cells; andadministering these conjugated antibodies to the patient; wherein saidanti-cancer antibodies are administered in admixture with apharmaceutically acceptable adjuvant, for example normal saline, a lipidemulsion, albumen, phosphate buffered saline or the like and areadministered in an amount effective to mediate treatment of saidcancerous disease, for example with a range of about 1 microgram per milto about 1 gram per mil. In a particular embodiment, the anti-cancerantibodies useful in either of the above outlined methods may be ahumanized antibody.

What is claimed is:
 1. A method for treating a patient suffering from acancerous disease comprising: administering to said patient anti-cancerantibodies or fragments thereof produced in accordance with a method forthe production of individually customized anti-cancer antibodies whichare useful in treating a cancerous disease, said antibodies including asubset of antibodies or fragments thereof characterized as beingcytotoxic against cells of a cancerous tissue, said subset beingessentially benign to non-cancerous cells; wherein one or moreantibodies or fragments thereof selected from said subset are placed inadmixture with a pharmaceutically acceptable adjuvant and areadministered in an amount effective to mediate treatment of saidcancerous disease; said one or more antibodies or fragments thereofbeing selected from the group consisting of a 1LN-8, 4BD-1, a 4BD-3, a4BD-6, a 4BD-9, a 4BD-13, a 4BD-18, a 4BD-20, a 4BD-25, a 4BD-26, a4BD-27, a 4BD-28, a 4BD-32, a 4BD-37, a 4BD-50, a 6BD-1, a 6BD-3, a6BD-5, a 6BD-11, a 6BD-25, a 7BD-7, a 7BD-12-1, a 7BD-12-2, a 7BD-13, a7BD-14, a 7BD-19, a 7BD-21, a 7BD-24, a 7BD-29, a 7BD-30, a 7BD-31, a7BDI-17, a 7BDI-58, a 7BDI-60, a 7BDI-62, a 5LAC2, a 5LAC4, a 5LAC20, a5LAC23, a H460-1, a H460-4, a H460-5, a H460-10, a H460-14, a H460-16-1,a H460-16-2, a H460-23 and a H460-27 monoclonal antibody or combinationsthereof.
 2. The method for treating a patient suffering from a cancerousdisease in accordance with claim 1, wherein said one or more antibodiesor fragments thereof selected from said subset are humanized.
 3. Themethod for treating a patient suffering from a cancerous disease inaccordance with claim 1 comprising: conjugating said subset ofantibodies or fragments thereof with a member selected from the groupconsisting of toxins, enzymes, radioactive compounds, and hematogenouscells; and administering conjugated antibodies or fragments thereof tosaid patient; wherein said conjugated antibodies are placed in admixturewith a pharmaceutically acceptable adjuvant and are administered in anamount effective to mediate treatment of said cancerous disease.
 4. Themethod of claim 3, wherein said one or more antibodies or fragmentsthereof selected from said subset are humanized.
 5. The method fortreating a patient suffering from a cancerous disease in accordance withclaim 1 wherein: the cytotoxicity of said antibodies or fragmentsthereof is mediated through antibody dependent cellular toxicity.
 6. Themethod for treating a patient suffering from a cancerous disease inaccordance with claim 1 wherein: the cytotoxicity of said antibodies orfragments thereof is mediated through complement dependent cellulartoxicity.
 7. The method for treating a patient suffering from acancerous disease in accordance with claim 1 wherein: the cytotoxicityof said antibodies or fragments thereof is mediated through catalyzingof the hydrolysis of cellular chemical bonds.
 8. The method for treatinga patient suffering from a cancerous disease in accordance with claim 1wherein: the cytotoxicity of said antibodies or fragments thereof ismediated through producing an immune response against putative cancerantigens residing on tumor cells.
 9. The method for treating a patientsuffering from a cancerous disease in accordance with claim 1 wherein:the cytotoxicity of said antibodies or fragments thereof is mediatedthrough targeting of cell membrane proteins to interfere with theirfunction.
 10. The method for treating a patient suffering from acancerous disease in accordance with claim 1 wherein: the cytotoxicityof said antibodies or fragments thereof is mediated through productionof a conformational change in a cellular protein effective to produce asignal to initiate cell-killing.
 11. The method for treating a patientsuffering from a cancerous disease in accordance with claim 1 wherein:said method of production utilizes a tissue sample containing cancerousand non-cancerous cells obtained from a particular individual.
 12. Amethod for treating a patient suffering from a cancerous diseasecomprising: administering to said patient anti-cancer antibodies orfragments thereof produced in accordance with a method for theproduction of individually customized anti-cancer antibodies which areuseful in treating a cancerous disease, said antibodies including asubset of antibodies or fragments thereof characterized as beingcytotoxic against cells of a cancerous tissue, said subset beingessentially benign to non-cancerous cells; wherein one or moreantibodies or fragments thereof selected from said subset are placed inadmixture with a pharmaceutically acceptable adjuvant and areadministered in an amount effective to mediate treatment of saidcancerous disease; said one or more antibodies or fragments thereofproduced by a hybridoma cell line having an ATCC Accession Numberselected from the group consisting of ( ) or combinations thereof. 13.The method for treating a patient suffering from a cancerous disease inaccordance with claim 12, wherein said one or more antibodies orfragments thereof selected from said subset are humanized.
 14. Themethod for treating a patient suffering from a cancerous disease inaccordance with claim 12 comprising: conjugating said subset ofantibodies or fragments thereof with a member selected from the groupconsisting of toxins, enzymes, radioactive compounds, and hematogenouscells; and administering conjugated antibodies or fragments thereof tosaid patient; wherein said conjugated antibodies are placed in admixturewith a pharmaceutically acceptable adjuvant and are administered in anamount effective to mediate treatment of said cancerous disease.
 15. Themethod of claim 14, wherein said one or more antibodies or fragmentsthereof selected from said subset are humanized.
 16. The method fortreating a patient suffering from a cancerous disease in accordance withclaim 12 wherein: the cytotoxicity of said antibodies or fragmentsthereof is mediated through antibody dependent cellular toxicity. 17.The method for treating a patient suffering from a cancerous disease inaccordance with claim 12 wherein: the cytotoxicity of said antibodies orfragments thereof is mediated through complement dependent cellulartoxicity.
 18. The method for treating a patient suffering from acancerous disease in accordance with claim 12 wherein: the cytotoxicityof said antibodies or fragments thereof is mediated through catalyzingof the hydrolysis of cellular chemical bonds.
 19. The method fortreating a patient suffering from a cancerous disease in accordance withclaim 12 wherein: the cytotoxicity of said antibodies or fragmentsthereof is mediated through producing an immune response againstputative cancer antigens residing on tumor cells.
 20. The method fortreating a patient suffering from a cancerous disease in accordance withclaim 12 wherein: the cytotoxicity of said antibodies or fragmentsthereof is mediated through targeting of cell membrane proteins tointerfere with their function.
 21. The method for treating a patientsuffering from a cancerous disease in accordance with claim 12 wherein:the cytotoxicity of said antibodies or fragments thereof is mediatedthrough production of a conformational change in a cellular proteineffective to produce a signal to initiate cell-killing.
 22. The methodfor treating a patient suffering from a cancerous disease in accordancewith claim 12 wherein: said method of production utilizes a tissuesample containing cancerous and non-cancerous cells obtained from aparticular individual.
 23. Anti-cancer antibodies or fragments thereofselected from the group consisting of a 1LN-8, 4BD-1, a 4BD-3, a 4BD-6,a 4BD-9, a 4BD-13, a 4BD-18, a 4BD-20, a 4BD-25, a 4BD-26, a 4BD-27, a4BD-28, a 4BD-32, a 4BD-37, a 4BD-50, a 6BD-1, a 6BD-3, a 6BD-5, a6BD-11, a 6BD-25, a 7BD-7, a 7BD-12-1, a 7BD-12-2, a 7BD-13, a 7BD-14, a7BD-19, a 7BD-21, a 7BD-24, a 7BD-29, a 7BD-30, a 7BD-31, a 7BDI-17, a7BDI-58, a 7BDI-60, a 7BDI-62, a 5LAC2, a 5LAC4, a 5LAC20, a 5LAC23, aH460-1, a H460-4, a H460-5, a H460-10, a H460-14, a H460-16-1, aH460-16-2, a H460-23 and a H460-27 monoclonal antibody or combinationsthereof.
 24. Anti-cancer antibodies or fragments thereof produced by ahybridoma cell line having an ATCC Accession Number selected from thegroup consisting of ( ).